Early experimental work suggested that the retina’s main role was to detect changes in brightness and contrast, namely working as a light detector, and that most of the complex computations in the visual system happened upstream in the brain. In reality, there is a growing wealth of literature indicating that the retina itself processes multiple channels of visual information (contrast, motion, orientation, etc.), making it much more complex than it originally appeared. For instance, there now appear to be over 20 types of retinal ganglion cells. To this end, the work in this thesis will focus on the identification and characterization of a single type of retinal ganglion cell in the mouse retina. In the first section of my results, I will show that this cell type, identified as the only GFP+ ganglion cell in the transgenic Hb9::eGFP retina, is a directionally selective ganglion cell (DSGC), that preferentially responds to objects moving upward through the visual field. This cell has a pronounced morphological asymmetry that helps it to synergistically (along with asymmetric inhibition) generate directionally selective responses. In the second results section, I will describe a novel phenomenon exhibited by Hb9+ DSGCs: Thanks to gap junction mediated signals, Hb9+ cells are able to anticipate moving stimuli and correct for lags that are inherent in visual signals generated by photoreceptors. In the third results section I will elucidate the mechanisms for the gap junction mediated anticipatory signals outlined in the second results section. Together, these results provide a significant advancement in our understanding of how the retina processes moving stimuli and provide a compelling example of how chemical and electrical synapses interact to allow for exquisite signal multiplexing.

Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

The Royal College of Surgeons (RCS) rat was used as a model of human retinal degenerative disease, and for studying the efficacy of cell transplanation treatments. In order to characterize the spatial vision of the RCS strain, the visual acutiy and contrast sensitivity of adult non-dystrophic RCS rats was measured. The acuity and contrast sensitivity of these rats was normal. The acuity of dystrophic RCS rats was alos characterized to determine how photoreceptor degeneration affects vision. These rats progressively lost visual acuity from one month of age until elevn months of age when they were judged to be blind. The degeneration of vision in these animals was more protacted than would be predicted from previous anatomical and electrophysiological measures. Subretinal transplantation of human-derived Retinal Pigment Epithelial (RPE) cells and human Schwann cells into the dystrophic RCS rat significantly delayed the loss of visual acuity. These studies show that cell transplantation may be a viable method of limiting loss of vision in humans with retinal degenerative blinding diseases. / vii, 77 leaves ; 29 cm.

The established model used for evaluating potential therapies for retinal disease has significant limitations. A new model is proposed to account for these limitations: the visual adaptation model. The visual adaptation model was developed to provide a novel approach for testing potential treatments for retinal disease, and the work in this thesis provides empirical support for this model. Specifically, we evaluated two potential therapies for retinal degenerative disease and examined their effects on vision and retinal anatomy. In addition, the profile of retinal reorganization and its functional correlates were examined in RCS rats and transgenic rats which express a rhodopsin mutation; however, immunohistological work targeted one specific line (S334ter-4). Collectively, these studies provide evidence that supports the retinal adaptation model. These studies also provide a novel view of retinal and visual function in retinal disease which should be considered when evaluating treatments involving retinal degeneration. / xvii, 205 leaves : ill. ; 29 cm. --

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Les effets de la marijuana, un médicament utilisé par l’homme depuis des millénaires, sur le système visuel sont peu connus. Une meilleure connaissance de la distribution du système endocannabinoïde (eCB) de la rétine pourrait expliquer comment cette drogue affecte la vision. Cette étude vise à caractériser la distribution du récepteur cannabinoïde CB1 (CB1R) et de l’enzyme de dégradation FAAH (“fatty acid amide hydrolase”) des ligands du CB1R dans la rétine du singe Vert (Chlorocebus sabaeus). De plus, elle vise à déterminer quelles sous-populations cellulaires de la rétine expriment ces composantes. La plupart des études à ce jour ont été conduites surtout sur les rongeurs et peu de travaux ont été réalisés chez le singe. Notre étude vient donc combler cette carence. Par le biais de méthodes immunohistochimiques, nous avons investigué la localisation du CB1R et de l’enzyme FAAH à différentes excentricités rétiniennes, de la fovéa centralis vers la périphérie. Nos résultats, en accord avec notre hypothèse de travail, démontrent que CB1R et FAAH sont exprimés à travers toute la rétine mais avec, cependant, des différences notoires. Au niveau de la couche des photorécepteurs, CB1R est exprimé préférentiellement dans les cônes et ce patron d’expression suit la distribution des photorécepteurs centre-périphérie. De plus, CB1R se retrouve surtout dans les pédicules des cônes de la couche plexiforme externe. CB1R et FAAH sont abondants dans les cellules bipolaires tant au centre qu’en périphérie. Le soma et l’axone des cellules ganglionnaires expriment aussi CB1R et FAAH. Ces données suggèrent que le système eCB est présent à travers toute la rétine du primate et pourrait expliquer les perturbations visuelles entrainées par la marijuana, telles la photosensibilité et la vision des couleurs. / The effects of marijuana, a drug that has been used by men for millennia, on the visual system are poorly understood. A better understanding of the distribution of the endocannabinoid system in the retina will help us explain how this drug affects vision. This study aims at characterizing the distribution of the endocannabinoid receptor CB1 (CB1R) and the enzyme degrading CB1R ligands, fatty acid amide hydrolase (FAAH) throughout the Green monkey retina (Chlorocebus sabaeus). In addition, it seeks to determine which sub-population of neurons expresses CB1R and the degrading enzyme FAAH. Most data on the endocannabinoid system have been acquired in rodents and studies on monkeys are rather scarce. We attempted to fill this void by using immunohistochemical methods to locate CB1R and FAAH at various eccentricities of the monkey retina, from the center to the far periphery. Our results, in agreement with our hypothesis, demonstrate that CB1R and FAAH are expressed throughout the retina. At the level of the photoreceptors, CB1R is expressed preferentially in cones rather than in rods, and this expression pattern follows the photoreceptors distribution. In the outer plexiform layer, CB1R immunoreactivity is predominantly concentrated in the cone pedicles. Although foveal cones are the main expressers of both CB1R and FAAH, these are also found in rod bipolar cells. The ganglion cell axons strongly express the CB1 receptor and the enzyme FAAH. These data suggest that the presence of CB1R throughout the retina may be responsible for the visual effects commonly reported by cannabis users, such as the increase in photosensitivity and alterations in color discrimination.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.

Cellular transplantation for photoreceptor replacement in retinal disease is limited by poor distribution, survival and integration of cells in vivo after standard delivery in saline vehicle. We were interested in addressing each of these barriers in order to improve transplant efficacy. To this end, we designed the first injectable biomaterial-based cell delivery vehicle to transplant adult stem cell progeny into the subretinal space of adult retina. A minimally-invasive and bio-resorbable blend of hyaluronan and methylcellulose (HAMC) was found to overcome cellular aggregation and non-contiguous distribution. The ability to direct stem cell differentiation toward a particular retinal lineage is another challenge facing clinical application. We showed that prospectively and clonally isolated multipotent mouse and human retinal stem cells (RSCs) could be directed toward a mature rod photoreceptor fate with the highest efficiency reported to date (>90%). Combinations of taurine and retinoic acid directed rod differentiation similar to rod development in vivo. RSC-derived rods exhibited morphology, protein and gene expression consistent with primary cultures of rods in vitro. When combined with the HAMC delivery vehicle, greater cell survival and improved integration of post-mitotic RSC-derived rods was observed in vivo compared to saline delivery. Improved donor rod survival was ascribed to the CD44 receptor – HAMC interaction in vitro and in vivo. Combined with HAMC delivery, disruption of the glial limiting membrane improved cell integration and resulted in the highest levels of integration of adult stem cell-derived rod photoreceptors relative to previous reports in the literature. Mature rod-lineage committed cells demonstrated higher integration potential compared to immature rods in this context. The integrated cells expressed Rhodopsin and elaborated outer segments. In the absence of the glial limiting membrane, rod integration depended on pro-survival signals from the environment. This work demonstrates that adult RSCs show promise for regenerative medicine strategies in the adult retina.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.

The main objective of this thesis is to investigate the intrinsic optical signals (IOSs) with an ultra-high resolution optical coherence tomography system (UHROCT). In order to study the retinal IOSs evoked by visible light, an UHROCT and an Electroretinogram (ERG) system was combined. An animal model (chicken retina) based on its retinal avascularity and cone dominance, was selected. Imaging the chicken retina with OCT resulted in high contrast, high resolution (~3μm axial and ~5 μm lateral resolution) 2D and 3D volumetric tomograms, in which all retina layers were clearly distinguishable. Using the combined UHROCT and ERG system to image IOSs from the chicken retina exposed to visible light (7ms green flash) resulted in highly reproducible IOS recordings from all retinal layers for the first time. All inner retinal layers showed an initial increase and subsequently a decrease in the intensity of the backreflected imaging light within the first 100 ms after the onset of the stimulus. Outer segments of the photoreceptors also showed a decrease in the backreflected imaging light within 100 ms after the onset of the flash. All retinal layers showed a strong decrease in the backreflected light within 150 to 175 ms after the onset of the flash. Imaging the pupil dynamics of the chicken with a modified combined UHROCT and ERG system showed that part of the strong negative IOSs observed in all retinal layers resulted from the vignetting of the imaging beam due to the light induced pupil constriction. Thorough analysis of the pupil dynamics acquired with UHROCT showed a time dependent effect of the anesthesia agent on pupil constriction. Further experiments to investigate an anesthesia effects on retinal function showed significant changes in ERG components. Statistical analysis showed that Isoflurane anesthesia severely affects the inner retinal response. In conclusion, it was hypothesized that the fast IOSs within ~50-100 ms after the onset of the visual stimulus originated from the neuronal tissue in the retina and are related to tissue optical property changes as a result of the electrical signal propagation in the light activated retina. Longer term decreases in backreflected light are likely due to pupil changes.